Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice / 中华烧伤杂志

Objective: To investigate the role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice. Methods: The experimental research methods were applied. Eighty-six C57BL/6J male mice (hereinafter briefly referred to as wild-type mice) aged 8-12 weeks were selected for the following experiments. Vγ4 T cells were isolated from axillary lymph nodes of five wild-type mice for the following experiments. Intraperitoneal injection of rapamycin for 42 mice was performed to establish rapamycin-treated mice model for the following experiments. Eighteen wild-type mice were divided into normal control group without any treatment, trauma only group, and trauma+CC chemokine ligand 20 (CCL20) inhibitor group according to the random number table (the same grouping method below), with 6 mice in each group. The full-thickness skin defect wound was made on the back of mice in the latter two groups (the same wound model below), and mice in trauma+CCL20 inhibitor group were continuously injected subcutaneously with CCL20 inhibitor at the wound edge for 3 days after injury. Another 6 rapamycin-treated mice were used to establish wound model as rapamycin+trauma group. On post injury day (PID) 3, the epidermal cells of the skin tissue around the wound of each trauma mice were extracted by enzyme digestion, and the percentage of Vγ4 T cells in the epidermal cells was detected by flow cytometry. In normal control group, the epidermal cells of the normal skin tissue in the back of mice were taken at the appropriate time point for detection as above. Five wild-type mice were used to establish wound models. On PID 3, the epidermal cells were extracted from the skin tissue around the wound. The cell populations were divided into Vγ4 T cells, Vγ3 T cells, and γδ negative cells by fluorescence-activated cell sorter, which were set as Vγ4 T cell group, Vγ3 T cell group, and γδ negative cell group (with cells in each group being mixed with B16 mouse melanoma cells), respectively. B16 mouse melanoma cells were used as melanoma cell control group. The expression of interleukin-22 (IL-22) mRNA in cells of each group was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with the number of samples being 6. Thirty rapamycin-treated mice were used to establish wound models, which were divided into Vγ4 T cell only group and Vγ4 T cell+IL-22 inhibitor group performed with corresponding injections and rapamycin control group injected with phosphate buffer solution (PBS) immediately after injury, with 10 mice in each group. Another 10 wild-type mice were taken to establish wound models and injected with PBS as wild-type control group. Mice in each group were injected continuously for 6 days. The percentage of wound area of mice in the four groups was calculated on PID 1, 2, 3, 4, 5, and 6 after injection on the same day. Six wild-type mice and 6 rapamycin-treated mice were taken respectively to establish wound models as wild-type group and rapamycin group. On PID 3, the mRNA and protein expressions of IL-22 and CCL20 in the peri-wound epidermis tissue of mice in the two groups were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively. The Vγ4 T cells were divided into normal control group without any treatment and rapamycin-treated rapamycin group. After being cultured for 24 hours, the mRNA and protein expressions of IL-22 of cells in the two groups were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively, with the number of samples being 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, Bonferroni method, Kruskal-Wallis H test, and Wilcoxon rank sum test. Results: The percentage of Vγ4 T cells in the epidermal cells of the skin tissue around the wound of mice in trauma only group on PID 3 was 0.66% (0.52%, 0.81%), which was significantly higher than 0.09% (0.04%, 0.14%) in the epidermal cells of the normal skin tissue of mice in normal control group (Z=4.31, P<0.01). The percentages of Vγ4 T cells in the epidermal cells of the skin tissue around the wound of mice in rapamycin+trauma group and trauma+CCL20 inhibitor group on PID 3 were 0.25% (0.16%, 0.37%) and 0.24% (0.17%, 0.35%), respectively, which were significantly lower than that in trauma only group (with Z values of 2.27 and 2.25, respectively, P<0.05). The mRNA expression level of IL-22 of cells in Vγ4 T cell group was significantly higher than that in Vγ3 T cell group, γδ negative cell group, and melanoma cell control group (with Z values of 2.96, 2.45, and 3.41, respectively, P<0.05 or P<0.01). Compared with that in wild-type control group, the percentage of wound area of mice in rapamycin control group increased significantly on PID 1-6 (P<0.01), the percentage of wound area of mice in Vγ4 T cell+IL-22 inhibitor group increased significantly on PID 1 and PID 3-6 (P<0.05 or P<0.01). Compared with that in rapamycin control group, the percentage of wound area of mice in Vγ4 T cell only group decreased significantly on PID 1-6 (P<0.05 or P<0.01). Compared with that in Vγ4 T cell only group, the percentage of wound area of mice in Vγ4 T cell+IL-22 inhibitor group increased significantly on PID 3-6 (P<0.05 or P<0.01). On PID 3, compared with those in wild-type group, the expression levels of IL-22 protein and mRNA (with t values of -7.82 and -5.04, respectively, P<0.01) and CCL20 protein and mRNA (with t values of -7.12 and -5.73, respectively, P<0.01) were decreased significantly in the peri-wound epidermis tissue of mice in rapamycin group. After being cultured for 24 hours, the expression levels of IL-22 protein and mRNA in Vγ4 T cells in rapamycin group were significantly lower than those in normal control group (with t values of -7.75 and -6.04, respectively, P<0.01). Conclusions: In mice with full-thickness skin defects, rapamycin may impair the CCL20 chemotactic system by inhibiting the expression of CCL20, leading to a decrease in the recruitment of Vγ4 T cells to the epidermis, and at the same time inhibit the secretion of IL-22 by Vγ4 T cells, thereby slowing the wound healing rate.

TCMATCOV--a bioinformatics platform to predict efficacy of TCM against COVID-19 / 中国中药杂志

There is urgent need to discover effective traditional Chinese medicine(TCM) for treating coronavirus disease 2019(COVID-19). The development of a bioinformatic tool is beneficial to predict the efficacy of TCM against COVID-19. Here we deve-loped a prediction platform TCMATCOV to predict the efficacy of the anti-coronavirus pneumonia effect of TCM, based on the interaction network imitating the disease network of COVID-19. This COVID-19 network model was constructed by protein-protein interactions of differentially expressed genes in mouse pneumonia caused by SARS-CoV and cytokines specifically up-regulated by COVID-19. TCMATCOV adopted quantitative evaluation algorithm of disease network disturbance after multi-target drug attack to predict potential drug effects. Based on the TCMATCOV platform, 106 TCM were calculated and predicted. Among them, the TCM with a high disturbance score account for a high proportion of the classic anti-COVID-19 prescriptions used by clinicians, suggesting that TCMATCOV has a good prediction ability to discover the effective TCM. The five flavors of Chinese medicine with a disturbance score greater than 1 are mainly spicy and bitter. The main meridian of these TCM is lung, heart, spleen, liver, and stomach meridian. The TCM related with QI and warm TCM have higher disturbance score. As a prediction tool for anti-COVID-19 TCM prescription, TCMATCOV platform possesses the potential to discovery possible effective TCM against COVID-19.

Identification and characterization of a novel group of natural antisense transcripts from RNA1.2 gene locus of human cytomegalovirus / 中华医学杂志(英文版)

Background@#Natural anti-sense transcripts (NATs), which are transcribed from the complementary DNA strand of annotated genes, exert regulatory function of gene expression. Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus (HCMV) genome, whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated. In this study, the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain.@*Methods@#Strand-specific high-through RNA-sequencing (RNA-seq) was performed to find possible anti-sense transcripts (ASTs). For analyzing and visualization of RNA-seq data sets, Integrative Genomics Viewer software was applied. To confirm these possibilities, Northern blotting and rapid amplification of cDNA ends (RACE) were used.@*Results@#Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN. At least three HCMV NATs, named RNA1.2 AST 1, RNA1.2 AST2, and RNA1.2 AST3, were characterized by Northern blotting and RACE analyses. These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection. The 5′- and 3′-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene.@*Conclusion@#A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA1.2 gene region.

Identification and characterization of a novel group of natural anti-sense transcripts from RNA1.2 gene locus of human cytomegalovirus / 中华医学杂志(英文版)

BACKGROUND@#Natural anti-sense transcripts (NATs), which are transcribed from the complementary DNA strand of annotated genes, exert regulatory function of gene expression. Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus (HCMV) genome, whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated. In this study, the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain.@*METHODS@#Strand-specific high-through RNA-sequencing (RNA-seq) was performed to find possible anti-sense transcripts (ASTs). For analyzing and visualization of RNA-seq data sets, Integrative Genomics Viewer software was applied. To confirm these possibilities, Northern blotting and rapid amplification of cDNA ends (RACE) were used.@*RESULTS@#Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN. At least three HCMV NATs, named RNA1.2 AST 1, RNA1.2 AST2, and RNA1.2 AST3, were characterized by Northern blotting and RACE analyses. These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection. The 5'- and 3'-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene.@*CONCLUSION@#A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA1.2 gene region.